The invention is in the field of molecular biology.
The determination of nucleic acid base sequences is important for both research and diagnostics. Many techniques for determining nucleic acid base sequences have been developed over the years, e.g., controlled chemical degradation (Maxim and Gilbert, Proceedings of the National Academy of Sciences USA 74: 560-564 (1977), 2xe2x80x23xe2x80x2 dideoxy chain termination method (Sanger et al. Proceedings of the National Academy of Sciences USA 74: 5463-5467 (1977). A variation of the technique of chain termination sequencing is known as single base extension or xe2x80x9cmini-sequencingxe2x80x9d is performed when nucleic acid base sequence information is required for only a single base site adjacent to the 3xe2x80x2 terminus oligonucleotide primer. The technique of single base extension is described in U.S. Pat. No. 5,856,092 and Syvanen et al. Genomics 8, 684-692(1990).A problem with single base extension techniques is the difficulty associated with identifying the single base extension product, particularly in an electrophoresis . Variations in the signal produced by the single base extension product, e.g., as detected by an electrophoresis apparatus, maybe the result of variations in signal produced by differences between oligonucleotide primers. The problems associated with identifying single base extension products become particularly troublesome in multiplexed single base extension reactions. The inventors have provided methods, compositions, kits and software for ameliorating these problems associated with identifying single base extension reaction products.
One embodiment of the invention is a method of producing an oligonucleotide extended by a single nucleotide base. An oligonucleotide and an extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The reaction produces an oligonucleotide extended by a single base. The extended oligonucleotide may be used as a size standard for single base extension reactions.
Another embodiment of the invention is a method of producing a mixture of oligonucleotides extended by different single bases. An oligonucleotide, a first extension terminating nucleotide, and a second extension terminating nucleotide are mixed with an enzyme having terminal transferase activity. The first and second extension terminating nucleotides comprise different nucleotide bases and are labeled with different labels. The identity of the different extension terminating nucleotides (and hence the extended oligonucleotides) may be ascertained by reference to the specific label incorporated. Another embodiment of the invention is a method of identifying the reaction products of single nucleotide base extension reactions on a detection instrument, e.g., an automated fluorescence detecting electrophoresis system, such as an Applied Biosystems PRISM(copyright) 377, PRISM(copyright) 3700 or PRISM(copyright) 3100. An oligonucleotide extension product is produced by mixing an oligonucleotide with an extension terminating nucleotide and an enzyme having terminal transferase activity, e.g., a terminal transferase. The single base oligonucleotide extension product may be used as a standard for comparison with the reaction products of single base extension reactions produced using a DNA polymerase, e.g., a mini-sequencing reaction product. Single base oligonucleotide extension products produced by the enzyme having terminal transferase activity may be resolved on a detection instrument, e.g. an electrophoresis apparatus, so as to produce a signal indicative of the single base extension product standard. The signal may be used as a standard for comparison with signals produced by the reaction products of template-dependent single base extension reaction products.
Other embodiments of the invention are kits for performing one or more methods of the invention. Embodiments of the subject kits include kits that comprise a terminal transferase and one or more extension terminating nucleotides. The extension terminating nucleotides may be labeled with the detectable moieties, such as fluorescent dyes.
The term xe2x80x9cterminal transferasexe2x80x9d as used herein refers to an enzyme having terminal transferase activity, but not having significant DNA polymerase activity. The term xe2x80x9csignificantxe2x80x9d as used in reference to DNA polymerase activity means DNA polymerase activity sufficient to perform a polynucleotide extension reaction that is template dependent at level sufficient to produce detectable amounts of template-dependent oligonucleotide extension product from an oligonucleotide primed template. Examples of terminal transferases include E. coli terminal transferase, calf thymus terminal transferase, and the like. Terminal transferases are commercially available from many companies such as Aphonix, Finnzyrnes, MBI Fernentas, New England Biolabs, Promega, Panvera, Sigma Biochemicals, and Roche Molecular Biochemicals .
The term xe2x80x9cterminal transferase activityxe2x80x9d as used herein refers to the enzymatic catalysis as of a reaction in which nucleotide triphosphates (including extension terminating nucleotides) are covalently attached to the 3xe2x80x2 terminus of an oligonucleotide primer in a template independent manner. Thus, by mixing an enzyme having terminal transferase activity within oligonucleotide having a free 3xe2x80x2-OH (or functional equivalent to) and with a nucleotide triphosphate, one or more nucleotides are added to the 3xe2x80x2 prime terminus of the oligonucleotide, irrespective of the presence or absence of a template complementary to the oligonucleotide.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein, unless clearly indicated otherwise by context, broadly refers to a polymer of natural or synthetic nucleobases, or a combination of both. The backbone of the capture polynucleotide can be composed entirely of xe2x80x9cnativexe2x80x9d phosphodiester linkages, or it may contain one or modified linkages, such as one or more phosphorothioate, phosphoramidite or other modified linkages. As a specific example, a polynucleotide may be a peptide nucleic acid (PNA), which contains amide interlinkages. Additional examples of modified bases and backbones that can be used in conjunction with the invention, as well as methods for their synthesis can be found, for example, in U.S. Pat. No. 6,001,983; Uhlman and Peyman, 1990, Chemical Review 90(4):544-584; Goodchild, 1990, Bioconjugate Chem. 1(3):165-186; Egholm et al., 1992, J. Am. Chem. Soc. 114:1895-1897; Gryaznov et al., J. Am. Chem. Soc. 116:3143-3144, as well as the references cited in all of the above. Common modified or synthetic nucleobases of which polynucleotides may be composed include 3-methlyuracil, 5,6-dihydrouracil, 4-thiouracil, 5-bromouracil, 5thorouracil, 5-iodouracil, 6-dimethyl amino purine, 6-methyl amino purine, 2-amino purine, 2,6-diamino purine, 6-amino-8-bromo purine, inosine, 5-methyl cytosine, 7-deazaadenine, and 7-deaza guanosine. Additional non-limiting examples of modified or synthetic nucleobases of which the target nucleic acid may be composed can be found in Fasman, CRC PRACTICAL HANDBOOK OF BIOCHEMISTRY AND MOLECULAR BIOLOGY, 1985, pp. 385-392; Beilstein""s Handbuch der Organischen Chemie, Springer Verlag, Berlin and Chemical Abstracts, all of which provide references to publications describing the structures, properties and preparation of such nucleobases. The term xe2x80x9coligonucleotidexe2x80x9d as used herein includes oligonucleotides that comprise additional molecules (or atoms) that have been joined, either covalently or non-covalently, to an oligonucleotide. These additional molecules (or atoms) maybe attached to virtually any site on the oligonucleotide, provided the attachment does not prevent the oligonucleotide from being used as a substrate for the enzyme having terminal transferase activity used in a given embodiment of the subject methods. Examples of such additional molecules include mobility modifier compounds such as those described as the subject of U.S. Pat. Nos. 5,514,543, 5,777,096, 5,703,096 and 5,470,705.
The term xe2x80x9cextension terminating nucleotidexe2x80x9d as used herein refers to refers to an enzymatically-incorporable nucleotide or nucleotide analog in which the sugar moiety does not support incorporation of subsequent nucleotides or nucleotide analogs. Typical terminators are those in which the nucleobase is a purine, a 7-deaza-purine, a pyrimidine, a normal nucleobase or a common analog thereof and the sugar moiety is a pentose which includes a 3xe2x80x2-substituent that blocks further synthesis, such as a ddNTP. Substituents that block further synthesis include, but are not limited to, amino, deoxy, halogen, alkoxy and aryloxy groups. Exemplary terminators include, but are not limited to, those in which the sugar-phosphate ester moiety is 3xe2x80x2-(C1-C6)alkylribose-5xe2x80x2-triphosphate, 2xe2x80x2-deoxy-3xe2x80x2-(C1-C6)alkylribose-5xe2x80x2-triphosphate, 2xe2x80x2-deoxy-3xe2x80x2-(C1-C6)alkoxyribose-5-triphosphate, 2xe2x80x2-deoxy-3xe2x80x2-(C5-C14)aryloxyribose-5xe2x80x2-triphosphate, 2xe2x80x2-deoxy-3xe2x80x2-haloribose-5xe2x80x2-triphosphate, 2xe2x80x2-deoxy-3xe2x80x2-aminoribose-5xe2x80x2-triphosphate, 2xe2x80x2,3xe2x80x2-dideoxyribose-5xe2x80x2-triphosphate or 2xe2x80x2,3xe2x80x2-didehydroribose-5xe2x80x2-triphosphate.
The term xe2x80x9cdetection instrumentxe2x80x9d as used herein refers to an analytical instrument capable of analyzing polynucleotides based on the size (or weight) of the polynucleotide. Examples of such detection instruments to include, but are not limited to, electrophoresis instruments (included automated DNA sequencers such as the Applied Biosystems ABI PRISM 377, ABI PRISM 310, ABI PRISM 3100, and ABI PRISM 3700) and mass spectragraphs, HPLC, and the like.
The term xe2x80x9cresolvedxe2x80x9d as used herein with respect to a detection instrument refers to detection of a specific signal indicative of a polynucleotide by the instrument. A polynucleotide that is said to be resolved by the instrument may be, but is not necessarily, separated or purified, from other polynucleotides in the mixture.
The term xe2x80x9cDNA polymerasexe2x80x9d as used herein refers to an enzyme capable of catalyzing in a template dependent manner the addition of nucleotide triphosphates to the 3xe2x80x2 terminus of an oligonucleotide that is hybridized to a complementary template.
The term xe2x80x9clabelxe2x80x9d as used, refers to a detectable moiety that maybe attached to a nucleotide in such a way as to permit the addition of the nucleotide (bearing the moiety) to the 3xe2x80x2 terminus of an oligonucleotide in a reaction catalyzed by a DNA polymerase. Detectable moieties produce a distinctive signal indicative of the presence of the moiety. Examples of detectable moieties may be fluorescent dyes, chromophores, chemiluminescent compounds, purified isotopes, and the like.
The term xe2x80x9csingle base extension reactionxe2x80x9d (or also xe2x80x9ctemplate-dependent single base extension reactionxe2x80x9d) refers to a method of determining the identity of a nucleotide base at a specific location on a polynucleotide template by extending an oligonucleotide hybridized to the template or single base at the 3xe2x80x2 position in a reaction catalyzed by a DNA polymerase. The extension of the oligonucleotide is by only a single base (as opposed to multiple bases) may be achieved by catalyzing the extension in the presence of one or more extension terminating nucleotides and in the absence of extendable nucleotides, e.g., 2xe2x80x2 deoxynucleotide triphosphates. The extension terminating nucleotides maybe differentially labeled so as to easily identified which bases been incorporated into the oligonucleotide. Thus, the 3xe2x80x2 position is extended by only a single base in a template dependent manner. Another method of achieving single base extension is by adding a single extendable nucleotide so that either extension with a lack of extension is detected. Descriptions of various methods of single base extension reactions can be found in U.S. Pat. No. 5,856,092 and Syvanen et al. Genomics 8, 684-692(1990). The oligonucleotide that has been extended by single base in a single base extension reaction in a template dependent manner (also referred to as xe2x80x9ctemplate-dependent single base extension reaction productsxe2x80x9d) is said to be a xe2x80x9csingle base extension reaction products.xe2x80x9d